Compounds and methods for modulation of estrogen receptors

ABSTRACT

Compounds that modulate gene expression through the estrogen receptor (ER) are disclosed having the following structure, as well as pharmaceutical compositions containing the same:                    
     wherein R 1 , R 2 , R 3 , n and p are as defined here. Methods are also disclosed for modulating ER in cells and/or tissues expressing the same, such as bone, breast, prostate, uterus, CNS or the cardiovascular system. Methods for treating estrogen-related conditions are also disclosed, including conditions such as is breast cancer, osteoporosis, endometriosis, cardiovascular disease, hypercholesterolemia, prostatic hypertrophy, prostatic carcinomas, obesity, hot flashes, skin effects, mood swings, memory loss, and adverse reproductive effects associated with exposure to environmental chemicals or natural hormonal imbalances.

CROSS-REFERENCE TO RELATED APPLICATION

This application is (i) a continuation-in-part of International Application No. PCT/US99/31290, filed Dec. 30, 1999, published under PCT Article 21(2) in English as International Publication No. WO 00/39120, which claims the benefit of U.S. Provisional Application No. 60/114,472, filed Dec. 30, 1998 and (ii) a continuation-in-part of U.S. application Ser. No. 09/475,776 filed Dec. 30, 1999, now abandoned, which claims the benefit of U.S. Provisional Application No. 60/114,472, filed Dec. 30, 1998.

TECHNICAL FIELD

This invention is generally directed to estrogen antagonists and agonists, and to compounds for inhibiting cytokines, as well as to pharmaceutical compositions and methods related thereto.

BACKGROUND OF THE INVENTION

The estrogen hormone has a broad spectrum of effects on tissues in both females and males. Many of these biological effects are positive, including maintenance of bone density, cardiovascular protection, central nervous system (CNS) function, and the protection of organ systems from the effects of aging. However, in addition to its positive effects, estrogen also is a potent growth factor in the breast and endometrium that increases the risk of cancer.

Until recently, it was assumed that estrogen binds to a single estrogen receptor (ER) in cells. As discussed below, this simple view changed significantly when a second ER (ER-β) was cloned (with the original ER being renamed ER-α), and when co-factors that modulate the ER response were discovered. Ligands can bind to two different ERs which, in the presence of tissue-specific co-activators and/or co-repressors, bind to an estrogen response element in the regulatory region of genes or to other transcription factors. Given the complexity of ER signaling, along with the tissue-specific expression of ER-α and ER-β and its co-factors, it is now recognized that ER ligands can act as estrogen agonists and antagonists that mimic the positive effects, or block the negative effects, of estrogen in a tissue-specific manner. This has given rise to the discovery of an entirely new class of drugs, referred to as Selective Estrogen Receptor Modulators or SERMs. These drugs have significant potential for the prevention and/or treatment of cancer and osteoporosis, as well as cardiovascular diseases and neurodegenerative diseases such as Alzheimer's disease.

Bone-resorbing diseases, such as osteoporosis, are debilitating conditions which affect a wide population, and to which there is only limited treatment. For example, osteoporosis affects about 50% of women, and about 10% of men, over the age of 50 in the United States. In individuals with osteoporosis, increased loss of bone mass results in fragile bones and, as a result, increased risk of bone fractures. Other bone-resorption diseases, such as Paget's disease and metastatic bone cancer, present similar symptoms.

Bone is a living tissue which contains several different types of cells. In healthy individuals, the amount of bone made by the osteoblastic cells is balanced by the amount of bone removed or resorbed by the osteoclastic cells. In individuals suffering from a bone-resorbing disease, there is an imbalance in the function of these two types of cells. Perhaps the most well known example of such an imbalance is the rapid increase in bone resorption experienced by postmenopausal women. Such accelerated bone lose is attributed to estrogen deficiency associated with menopause. However, the mechanism of how the loss of estrogen results in increased bone resorption has long been debated.

Recently, investigators have suggested that an increase in bone-resorbing cytokines, such as interleukin-1 (IL-1) and tumor necrosis factor (TNF), may be responsible for postmenopausal bone loss (Kimble et al., J. Biol. Chem. 271:28890-28897, 1996), and that inhibitors of these cytokines can partially diminish bone loss following ovariectomy in rodents (Pacifici, J. Bone Miner Res. 11:1043-1051, 1996). Further, discontinuation of estrogen has been reported to lead to an increase in IL-6 secretion by murine bone marrow and bone cells (Girasole et al., J. Clin. Invest. 89:883-891, 1992; Jilka et al., Science 257:88-91, 1992; Kimble et al., Endocrinology 136:3054-3061, 1995; Passseri et al., Endocrinology 133:822-828, 1993), antibodies against IL-6 can inhibit the increase in osteoclast precursors occurring in estrogen-depleted mice (Girasole et al, supra), and bone loss following ovariectomy does not occur in transgenic mice lacking IL-6 (Poli et al., EMBO J. 13:1189-1196, 1994).

Existing treatments for slowing bone loss generally involves administration of compounds such as estrogen, bisphosphonates, calcitonin, and raloxifene. These compounds, however, are generally used for long-term treatments, and have undesirable side effects. Further, such treatments are typically directed to the activity of mature osteoclasts, rather than reducing their formation. For example, estrogen induces the apoptosis of osteoclasts, while calcitonin causes the osteoclasts to shrink and detach from the surface of the bone (Hughes et al., Nat. Med. 2:1132-1136, 1996; (Jilka et al., Exp. Hematol. 23:500-506, 1995). Similarly, bisphosphonates decrease osteoclast activity, change their morphology, and increase the apoptosis of osteoclasts (Parfitt et al., J. Bone Miner 11:150-159, 1996; Suzuki et al., Endocrinology 137:4685-4690, 1996).

Cytokines are also believed to play an important role in a variety of cancers. For example, in the context of prostate cancer, researchers have shown IL-6 to be an autocrine/paracrine growth factor (Seigall et al., Cancer Res. 50:7786, 1999), to enhance survival of tumors (Okamoto et al., Cancer Res. 57:141-146, 1997), and that neutralizing IL-6 antibodies reduce cell proliferation (Okamoto et al., Endocrinology 138:5071-5073, 1997; Borsellino et al., Proc. Annu. Meet. Am. Assoc. Cancer Res. 37:A2801, 1996). Similar results have been reported for IL-6 with regard to multiple myeloma (Martinez-Maza et al., Res. Immunol. 143:764-769, 1992; Kawano et al., Blood 73:517-526, 1989; Zhang et al., Blood 74:11-13, 1989; Garrett et al., Bone 20:515-520, 1997; and Klein et al., Blood 78:1198-12-4, 1991), renal cell carcinoma (Koo et al., Cancer Immunol. 35:97-105, 1992; Tsukamoto et al., J. Urol. 148:1778-1782, 1992; and Weissglas et al., Endocrinology 138:1879-1885, 1997), and cervical carcinoma (Estuce et al., Gynecol. Oncol. 50:15-19, 1993; Tartour et al., Cancer Res. 54:6243-6248, 1994; and Iglesias et al., Am. J. Pathology 146:944-952, 1995).

Furthermore, IL-6 is also believed to be involved in arthritis, particularly in adjuvant-, collagen- and antigen-induced arthritis (Alonzi et al., J. Exp. Med. 187:146-148, 1998; Ohshima et al., Proc. Natl. Acad. Sci. USA 95:8222-8226, 1998; and Leisten et al., Clin. Immunol. Immunopathol 56:108-115, 1990), and anti-IL-6 antibodies have been reported for treatment of arthritis (Wendling et al., J. Rheumatol. 20:259-262, 1993). In addition, estrogen has been shown to induce suppression of experimental autoimmune encephalomyelitis and collagen-induced arthritis in mice (Jansson et al., Neuroimmunol. 53:203-207, 1994).

As noted above, it had previously been assumed that estrogen binds to a single estrogen receptor (ER) in cells, causing conformational changes that result in release from heat shock proteins and binding of the receptor as a dimer to the so-called estrogen response element in the promoter region of a variety of genes. Further, pharmacologists have generally believed that non-steroidal small molecule ligands compete for binding of estrogen to ER, acting as either antagonists or agonists in each tissue where the estrogen receptor is expressed. Thus, such ligands have traditionally been classified as either pure antagonists or agonists. This is no longer believed to be correct.

Rather, it is now known that estrogen modulates cellular pharmacology through gene expression, and that the estrogen effect is mediated by estrogen receptors. As noted above, there are currently two estrogen receptors, ER-α and ER-β. The effect of estrogen receptor on gene regulation can be mediated by a direct binding of ER to the estrogen response element (ERE)—“classical pathway” (Jeltsch et al., Nucleic Acids Res. 15:1401-1414, 1987; Bodine et al., Endocrinology 139:2048-2057, 1998), binding of ER to other transcription factors such as NF-κB, C/EBP-β or AP-1—“non-classcial pathway” (Stein et al., Mol. Cell Biol. 15:4971-4979, 1995; Paech et al., Science 277:1508-1510, 1997; Duan et al., Endocrinology 139:1981-1990, 1998), and through non-genomic effects involving ion channel receptors (Watters et al., Endocrinology 138:4030-4033, 1997; Improta-Brears et al., Proc. Natl. Acad. Sci. USA 96:4686-4691, 1999; Gu et al., Endocrinology 140:660-666, 1999; Beyer et al., Eur. J. Neurosci. 10:255-262, 1998).

Progress over the last few years has shown that ER associates with co-activators (e.g., SRC-1, CBP and SRA) and co-repressors (e.g., SMRT and N-CoR), which also modulate the transcriptional activity of ER in a tissue-specific and ligand-specific manner. In addition, evidence now suggests that the majority of estrogen-regulated genes do not have a classical estrogen response element. In such cases, ER interacts with the transcription factors critical for regulation of these genes. Transcription factors known to be modulated in their activity by ER include, for example, AP-1, NF-κB, C/EBP and Sp-1.

Given the complexity of ER signaling, as well as the various types of tissue that express ER and its co-factors, it is now believed that ER ligands can no longer simply be classified as either pure antagonists or agonists. Therefore, the term “selective estrogen receptor modulator” (SERM) has been coined. SERMs bind to ER, but may act as an agonist or antagonist of estrogen in different tissues and on different genes. For example, two of the most well known drugs that behave as SERMs are Tamoxifen and Raloxifene. Studies with these two compounds, as well as other SERMs now in development, have demonstrated that the affinity of a SERM for its receptor in many cases does not correlate with its biological activity. Therefore, ligand-binding assays traditionally used in screening for novel ER modulators have not distinguished between tissue-selectivity and agonist/antagonist behavior.

More recently, a second estrogen receptor, ER-β, has been identified and cloned (Katzenellenbogen and Korach Endocrinology 138, 861-2 (1997); Kuiper et al., Proc. Natl. Acad. Sci. USA 93, 5925-5930, 1996; Mosselman et al., FEBS Lett. 392, 49-53, 1996). ER-β, and the classical ER renamed ER-α, have significantly different amino acid sequences in the ligand binding domain and carboxy-terminal transactivation domains (˜56% amino acid identity), and only 20% homology in their amino-terminal transactivation domain. This suggests that some ligands may have higher affinity to one receptor over the other. Further, ligand-dependent conformational changes of the two receptors, and interaction with co-factors, will result in very different biological actions of a single ligand. In other words, a ligand that acts as an agonist on ER-α may very well serve as an antagonist on ER-β. An example of such behavior has been described by Paech et al. (Science 277, 1508-1510, 1997). In that paper, estrogen is reported to activate an AP-1 site in the presence of ER-α, but to inhibit the same site in the presence of ER-β. In contrast, Raloxifene (Eli Lilly & Co.) and Tamoxifen and ICI-182,780 (Zeneca Pharmaceuticals) stimulate the AP-1 site through ER-β, but inhibit this site in the presence of ER-α. Another example has been described by Sun et al. (Endocrinology 140, 800-4, 1999). In this paper, the R,R-enantiomer of a tetrahydrochrysene is reported to be an agonist on ER-α, but a complete antagonist on ER-β, while the S,S-enantiomer is an agonist on both receptors.

Furthermore, ER-α and ER-β have both overlapping and different tissue distributions, as analyzed predominantly by RT-PCR or in-situ hybridization due to a lack of good ER-β antibodies. Some of these results, however, are controversial, which may be attributable to the method used for measuring ER, the species analyzed (rat, mouse, human) and/or the differentiation state of isolated primary cells. Very often tissues express both ER-α and ER-β, but the receptors are localized in different cell types. In addition, some tissues (such as kidney) contain exclusively ER-α, while other tissues (such as uterus, pituitary and epidymis) show a great predominance of ER-α (Couse et al., Endocrinology 138, 4613-4621, 1997; Kuiper et al., Endocrinology 138, 863-870, 1997). In contrast, tissues expressing high levels of ER-β include prostate, testis, ovaries and certain areas of the brain (Brandenberger et al., J. Clin. Endocrinol. Metab. 83, 1025-8, 1998; Enmark et al., J. Clinic. Endocrinol. Metabol. 82, 4258-4265, 1997; Laflamme et al., J. Neurobiol. 36, 357-78, 1998; Sar and Welsch, Endocrinology 140, 963-71, 1999; Shughrue et al., Endocrinology 138, 5649-52, 1997a; Shughrue et al., J. Comp. Neurol. 388, 507-25, 1997b).

The development of ER-α (Korach, Science 266, 1524-1527, 1994) and ER-β (Krege et al., Proc. Natl. Acad Sci. USA 95, 15677-82, 1998) knockout mice further demonstrate that ER-β has different functions in different tissues. For example, ER-α knockout mice (male and female) are infertile, females do not display sexual receptivity and males do not have typical male-aggressive behavior (Cooke et al., Biol. Reprod. 59, 470-5, 1998; Das et al., Proc. Natl. Acad. Sci. USA 94, 12786-12791, 1997; Korach, 1994; Ogawa et al., Proc. Natl. Acad. Sci. USA 94, 1476-81, 1997; Rissman et al., Endocrinology 138, 507-10, 1997a; Rissman et al., Horm. Behav. 31, 232-243, 1997b). Further, the brains of these animals still respond to estrogen in a pattern that is similar to that of wild type animals (Shughrue et al., Proc. Natl. Acad. Sci. USA 94, 11008-12, 1997c), and estrogen still inhibits vascular injury caused by mechanical damage (Iafrati et al., Nature Med. 3, 545-8, 1997). In contrast, mice lacking the ER-β develop normally, are fertile and exhibit normal sexual behavior, but have fewer and smaller litters than wild-type mice (Krege et al., 1998), have normal breast development and lactate normally. The reduction in fertility is believed to be the result of reduced ovarian efficiency, and ER-β is the predominant form of ER in the ovary, being localized in the granulosa cells of maturing follicles.

In summary, compounds which serve as estrogen antagonists or agonists have long been recognized for their significant pharmaceutical utility in the treatment of a wide variety of estrogen-related conditions, including conditions related to the brain, bone, cardiovascular system, skin, hair follicles, immune system, bladder and prostate (Barkhem et al., Mol. Pharmacol. 54, 105-12, 1998; Farhat et al., FASEB J. 10, 615-624, 1996; Gustafsson, Chem. Biol. 2, 508-11, 1998; Sun et al., 1999; Tremblay et al., Endocrinology 139, 111-118, 1998; Turner et al., Endocrinology 139, 3712-20, 1998). In addition, a variety of breast and non-breast cancer cells have been described to express ER, and serve as the target tissue for specific estrogen antagonists (Brandenberger et al., 1998; Clinton and Hua, Crit. Rev. Oncol. Hematol. 25, 1-9, 1997; Hata et al., Oncology 55 Suppl 1, 35-44, 1998; Rohlff et al., Prostate 37, 51-9, 1998; Simpson et al., J. Steroid Biochem Mol Biol 64, 137-45, 1998; Yamashita et al., Oncology 55 Suppl 1, 17-22, 1998).

In recent years a number of both steroidal and nonsteroidal compounds which interact with ER have been developed. For example, Tamoxifen was originally developed as an anti-estrogen and used for the treatment of breast cancer, but more recently has been found to act as a partial estrogen agonist in the uterus, bone and cardiovascular system. Raloxifene is another compound that has been proposed as a SERM, and has been approved for treatment of osteoporosis.

Analogs of Raloxifene have also been reported (Grese et al., J. Med. Chem. 40:146-167, 1997).

As for coumarin-based compounds, a number of structures have been proposed, including the following: Roa et al., Synthesis 887-888, 1981; Buu-Hoi et al., J. Org. Chem. 19:1548-1552, 1954; Gupta et al., Indian J. Exp. Biol. 23:638-640, 1985; Published PCT Application No. WO 96/31206; Verma et al., Indian J. Chem. 32B:239-243, 1993; Lednicer et al., J. Med. Chem. 8:725-726, 1965; Micheli et al., Steroids 5:321-335, 1962; Brandt et al., Int. J. Quantum Chemistry: Quantum Biol. Symposia 13:155-165, 1986; Wani et al., J. Med. Chem. 18:982-985, 1975; Pollard et al., Steroids 11:897-907, 1968.

Accordingly, there is a need in the art for estrogen antagonists and agonists generally, and more specifically for compounds that inhibit cytokines, particularly IL-6, including pharmaceutical compositions containing such compounds as well as methods relating to the use thereof The present invention fulfills these needs, and provides other related advantages.

SUMMARY OF THE INVENTION

In brief, the present invention is generally directed to estrogen antagonists and/or agonists, including pharmaceutical compositions containing the same, as well as to methods for treating estrogen-related conditions. Such conditions are more specifically discussed below, and generally include (but are not limited to) obesity, breast cancer, osteoporosis, endometriosis, cardiovascular disease, prostate cancer, menopausal syndromes, hair loss (alopecia), type-lI diabetes, Alzheimer's disease, urinary incontinence, GI tract conditions, spermatogenesis, vascular protection after injury, endometriosis, learning and memory, CNS effects, plasma lipid levels, acne, cataracts, hirsutism, other solid cancers (such as colon, lung, ovarian, melanoma, CNS, and renal), multiple myeloma, and lymphoma.

In a more specific embodiment, this invention is directed to compounds for inhibiting cytokines such as interleukin-6 (IL-6) and to methods relating to the treatment of conditions associated therewith, as well as pharmaceutical compositions containing one or more of the compounds of this invention. In this context, treating conditions associated with increased levels of cytokines include (but are not limited to) methods for treating cancer, arthritis and bone-resorbing diseases, particularly for reducing formation of osteoclasts and/or blocking cytokine production in the context of osteoporosis.

The compounds of this invention have the following general structure (I):

wherein R₁, R₂, R₃, n and p are as defined in the following detailed description, including stereoisomers, prodrugs and pharmaceutically acceptable salts thereof.

As noted above, the compounds of this invention have utility over a wide range of therapeutic and prophylactic applications, and may be used to treat a variety of diseases associated with bone resorption, as well as for treatment of cancer and arthritis. Such methods involve the administration of an effective amount of a compound of this invention, preferably in the form of a pharmaceutical composition, to an animal in need thereof (including a human).

In another embodiment, methods are disclosed for modulating cells and/or tissues that express ER by contacting the cell and/or tissue with an effective amount of a compound of structure (I). In one embodiment, the cell and/or tissue is that of bone, bladder, uterus, ovary, prostate, testis, epididymis, gastrointestinal (GI) tract, kidney, breast, heart, vessel wall, immune system, lung, eye, pituitary, hippocampus or hypothalamus.

In still a further embodiment, the present invention discloses methods for treating an estrogen-related condition by administering to an warm-blooded animal in need thereof an effective amount of a compound of structure (I) formulated as a pharmaceutical composition suitable for administration to the animal. In representative embodiments, the estrogen-related condition is breast cancer, osteoporosis, endometriosis, atherosclerosis, cardiovascular disease, hypercholesterolemia, prostatic hypertrophy, obesity, prostate cancer, menopausal syndromes, type-II diabetes, Alzheimer's disease, urinary incontinence, GI tract conditions, spermatogenesis, vascular protection after injury, endometriosis, learning and memory, CNS effects, plasma lipid levels, acne, hirsutism, other solid cancers (such as colon, lung, ovarian, melanoma, CNS, and renal), multiple myeloma, lymphoma, prostatic carcinomas, obesity, hot flashes, cataracts, skin effects, mood swings, memory loss, and/or adverse reproductive effects associated with exposure to environmental chemicals or natural hormonal imbalances.

These and other aspects of this invention will be evident upon reference to the following detailed description and attached drawings. To that end, various references are set forth herein which describe in more detail certain aspects of this invention, and which are hereby incorporated by reference in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B illustrate the activity of a representative compound of this invention to inhibit production of IL-6 and GM-CSF, respectively.

FIG. 2 illustrates the activity of a representative compound of this invention to inhibit IL-6 production in rheumatoid arthritis synoviocytes.

FIG. 3 illustrates the activity of a representative compound of this invention to inhibit breast cancer proliferation.

FIG. 4 illustrates the activity of a representative compound of this invention to inhibit a prostate cancer cell line.

DETAILED DESCRIPTION OF THE INVENTION

As mentioned above, this invention is generally directed to estrogen antagonists and agonists, and to compounds for inhibiting cytokines—that is, proteins produced by cells that alter the function of that cell or other cells—particularly interleukin-6 (IL-6). The compounds of this invention have the following general structure (I):

including stereoisomers, prodrugs and pharmaceutically acceptable salts thereof,

wherein:

n is 0, 1, 2, 3 or 4;

p is 0, 1 or 2;

R₁ is an unsubstituted or substituted C₆₋₁₂aryl, C₇₋₁₂arylalky, C₃₋₁₂heterocycle or C₄₋₁₆heterocyclealkyl;

R₂ is NR_(a)R_(b) wherein R_(a) and R_(b) are independently hydrogen, C₁₋₈alkyl, C₆₋₁₂aryl, or heterocycle, and wherein R_(a) and R_(b) are optionally substituted with up to three substituents independently selected from C₁₋₆allkyl, halogen, C₁₋₆alkoxy, hydroxy and carboxyl;

or R₂ is a heterocyclic ring of the following structure:

 wherein

m₁ and m₂ are independently 0, 1 or 2, and both of m₁ and m₂ are not 0,

A is CH₂, O, S or NH,

Z represents 0, 1, 2 or 3 heterocyclic ring substituents selected from halogen, C₁₋₈alkyl, C₆₋₁₂aryl, C₇₋₁₂arylalky, C₃₋₁₂heterocycle, or C₄₋₁₆heterocyclealkyl,

and wherein any hydrogen atom on the heterocyclic ring may, taken together with a hydrogen atom on an adjacent atom of the heterocylic ring, form a double bond;

R₃ is hydrogen, R₄, C(═O)R₄, C(═O)OR₄, CONHR₄, CONR₄R₅, or SO₂NR₅R₅;

R₄ and R₅ are independently C₁₋₈alkyl, C₆₋₁₂aryl, C₇₋₁₂aralkyl, or a five- or six-membered heterocycle containing up to two heteroatoms selected from O, NR₆ and S(O)_(q), wherein each of the above groups are optionally substituted with one to three substituents independently selected from R₇ and q is 0, 1 or 2;

R₆ is hydrogen or C₁₋₄alkyl; and

R₇ is hydrogen, halogen, hydroxy, C₁₋₆alkyl, C₁₋₄alkoxy, C₁₋₄acyloxy, C₁₋₄thio, C₁₋₄alkylsulfinyl, C₁₋₄alkylsulfonyl, (hydroxy)C₁₋₄alkyl, C₆₋₁₂aryl, C₇₋₁₂aralkyl, COOH, CN, CONHOR₈, SO₂NHR₈, NH₂, C₁₋₄alkylamino, C₁₋₄dialkylamino, NHSO₂R₈, NO₂, or a five- or six-membered heterocycle, where each occurrence of R₈ is independently C₁₋₆alkyl.

As used herein, a “C₆₋₁₂aryl” is an aromatic moiety containing from 6 to 12 carbon atoms. In one embodiment, the C₆₋₁₂aryl is selected from (but not limited to) phenyl, tetralinyl, and napthalenyl.

A “C₇₋₁₂aralkyl” is an arene containing from 7 to 12 carbon atoms, and has both aliphatic and aromatic units. In one embodiment, the C₇₋₁₂aralkyl is selected from (but not limited to) benzyl, ethylbenzyl (i.e., —(CH₂)₂phenyl), propylbenzyl and isobutylbenzyl.

A “C₃₋₁₂heterocycle” is a compound that contains a ring made up of more than one kind of atom, and which contains 3 to 12 carbon atoms, including (but not limited to) pyrrolyl, furanyl, thienyl, imidazolyl, oxazolyl, thiazolyl, pyrazolyl, pyrrolidinyl, pyridinyl, pyrimidinyl and purinyl.

A “C₄₋₁₆heterocyclealkyl” is a compound that contains a C₃₋₁₂heterocycle linked to a C₁₋₈alkyl.

A “C₁₋₈alkyl” is a straight chain or branched carbon chain containing from 1 to 8 carbon atoms, including (but not limited to) methyl, ethyl, and n-propyl. Similarly, a “C_(1-x)alkyl has the same meaning, but wherein “x” represents the number of carbon atoms less than eight, such as C₁₋₆alkyl.

A “substituted” C_(1-x)alkyl, C₆₋₁₂aryl, C₇₋₁₂aralkyl, C₃₋₁₂heterocycle, or C₄₋₁₆heterocyclealkyl moiety is a C_(1-x)alkyl, C₆₋₁₂aryl, C₇₋₁₂aralkyl, C₃₋₁₂heterocycle, or C₄₋₁₆heterocyclealkyl moiety having at least one hydrogen atom replaced with a substituent.

A “substituent” is a moiety selected from halogen, —OH, —R′, —OR′, —COOH, —COOR′, —COR′, —CONH₂, —NH₂, —NHR′, —NR′R′, —SH, —SR′, —SOOR′, —SOOH and —SOR′, where each occurrence of R′ is independently selected from an unsubstituted or substituted C₁₋₈alkyl, C₆₋₁₂aryl, C₇₋₁₂aralkyl, C₃₋₁₂heterocycle or C₄₋₁₆heterocyclealkyl.

A “halogen” is fluoro, chloro, bromo or iodo.

In one embodiment of this invention, A of the heterocyclic ring R₂ is CH₂, m₁, is 1, and m₂ is 0 or 1, as represented by the following structures (i) and (ii), respectively:

In structures (i) and (ii) above, it should be noted that the hydrogen atoms are not depicted in order to clarify that the optional Z substituent(s) may be on any atom of the heterocyclic ring, and that the point of attachment to structure (I) may be through a carbon or nitrogen atom.

Thus, in more specific embodiments of structures (i) and (ii) wherein Z is present, R₂ includes the following structures (iii) through (vi):

wherein Z is, for example, methyl.

In another embodiment, A of the heterocyclic ring R₂ is O or NH, m₁ is 1, and m₂ is 0 or 1, as represented by, for example, the following structures (vii) and (viii):

As with structures (i) and (ii) above, in structures (vii) and (viii) the hydrogen atoms are not depicted in order to clarif that the optional Z substituent(s) may be on any atom of the heterocyclic ring, and that the point of attachment to structure (I) may be through a carbon or nitrogen atom.

In addition to the above depicted structures, any hydrogen atom of the heterocyclic ring may be taken together with a hydrogen atom attached to an adjacent heterocyclic ring atom to form a double bond. For example, with regard to structure (vii) above, corresponding unsaturated analogs include the following structures (ix), (x) and (xi):

In one embodiment of this invention, R₁ is an unsubstituted or substituted phenyl, and the compounds of this invention have the following structure (II):

wherein X represents one or more optional substitutents as defined above, and R₂, R₃, n and p are as defined above.

In another embodiment, R₃ is hydrogen, as represented by structure (III):

wherein R₁, R₂, n and p are as defined above.

In a more specific embodiments of structures (II) and (III), representative compounds of this invention have the following structure (IV):

wherein A, X, Z, m₁, m₂, n and p are as defined above.

In a further embodiment of structure (IV), m₁, m₂ and p are 1, A is CH₂, the optional Z substituent is not present, and n is 2, and compounds of this invention have the following structure (V):

wherein X represents one or more optional substitutents as defined above.

In a more specific embodiment, X is either (a) not present or (b) represents a single substituent, such as a single substituent at the para position. Accordingly, representative compounds of this invention include (but are not limited to) compounds having the following structures (VIa) and (VIb):

wherein X in structure (VIb) represents a halogen, such as fluorine or chlorine.

The compounds of this invention may be made by one skilled in organic synthesis by known techniques, as well as by the synthetic routes disclosed herein. For example, representative compounds of this invention may be synthesized by the following general Reaction Scheme 1:

Reaction Scheme 1 yields compounds wherein R₃ is methyl or hydrogen, and R₂ is a heterocyclic ring as defined in structure (I). Further substitution at the R₃ position may be accomplished using an appropriately substituted phenol, or by subsequent conversion of the hydroxyl group (when R₃═H) using techniques known in the field of organic synthesis. Similarly, compounds of structure (I) wherein R₂ is NR_(a)R_(b) may be made by employing the corresponding amino chloride, R_(a)R_(b)N(CH₂)_(n)Cl, in place of the heterocyclic ring in the second-to-last step of Reaction Scheme 1.

More specifically, representative compounds of this invention (when R₃ is hydrogen and R₂ is piperid-1-yl) may be made by the following Reaction Scheme 2:

With regard to stereoisomers, the compounds of structure (I) may have chiral centers and may occur as recemates, reacemic mixtures and as individual enantiomers or diastereomers. All such isomeric forms are included within the present invention, including mixtures thereof. Furthermore, some of the crystalline forms of the compounds of structure (I) may exist as polymorphs, which are included in the present invention. In addition, some of the compounds of structure (I) may also form solvates with water or other organic solvents. Such solvates are similarly included within the scope of this invention.

Although not intending to be limited by the following theory, in the context of bone-resorbing diseases it is believed that the compounds of this invention function by blocking cytokine production and/or by inhibiting formation of osteoclasts. The cytokine IL-6 has previously been shown to be an important factor in inducing the formation of osteoclasts (Girasole et al., supra; Jilka et al. (1992), supra; Jilka et al. (1995), supra; Kimble et al. (1995), supra; Pacifici et al., supra; and Passeri et al., supra). Other investigators have shown that administration of the neutralizing antibody, antisense oligos, or the Sant 5 antagonist against IL-6, reduces the number of osteoclasts in trabecular bone of ovariectomized mice (Deylin et al., J. Bone Miner 13:393-399, 1998; Girasole et al., supra; Jilka et al. (1992), supra; and Schiller et al., Endocrinology 138:4567-4571, 1997), the ability of human giant cells to resorb dentine (Ohsaki et al., Endocrinology 131:2229-2234, 1993; and Reddy et al., J. Bone Min. Res. 9:753-757, 1994), and the formation of osteoclasts in normal human bone marrow culture. It has also been found that estrogen downregulates the IL-6 promoter activity by interactions between the estrogen receptor and the transcription factors NF-κB and C/EBPβ (Stein et al., Mol. Cell Biol. 15:4971-4979, 1995).

Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been suggested to play a role in the proliferation of osteoclastic precursor cells. In long term cultures of human or mouse bone marrow cells or peripheral blood cells, GM-CSF promotes the formation of osteoclastic cells (Kurihara et al., Blood 74:1295-1302, 1989; Lorenzo et al., J. Clin. Invest. 80:160-164, 1987; MacDonald et al., J. Bone Miner 1:227-233, 1986; and Shinar et al, Endocrinology 126:1728-1735, 1990). Bone marrow cells isolated from postmenopausal women, or women who discontinued estrogen therapy, expressed higher levels of GM-CSF than cells from premenopausal women (Bismar et al., J. Clin. Endocrinol. Metab. 80:3351-3355, 1995). Expression of GM-CSF has also been shown to be associated with the tissue distribution of bone-resorbing osteoclasts in patients with erosion of orthopedic implants (Al-Saffar et al., Anatomic Pathology 105:628-693, 1996).

In addition, it has been found that blocking the action of IL-6 with an anti-IL-6 antibody reduces the formation of osteoclast-like cells in the human bone cell co-culture system of Example 8. This system includes immortalized human osteoblasts and premonocytic cell line U937, which are co-cultured in the same tissue culture well. Under appropriate culture conditions the osteoblasts secret IL-6 which acts on the premonocytic cells causing their differentiation into osteoclast-like cells. Thus, the co-culture system more closely reflects the physiological environment of bone, and provides a functional readout for the efficacy of the compounds known to block the production of IL-6 and leading to osteoclast activation and formation. For example, estrogen was found to suppress the production of IL-6 in the co-culture system of Example 8.

Furthermore, an anti-GM-CSF antibody was found to reduce the formation of osteoclast-like cells, and estrogen reduced the production of GM-CSF in the co-culture system of Example 8. This demonstrates that estrogen may exert its inhibitory effect on osteoclast formation and function through blocking the production of IL-6 and GM-CSF. Therefore, compounds of this invention that block IL-6 and/or GM-CSF production, or inhibit formation and function of osteoclasts, or both, are useful in the treatment of bone-resorbing diseases.

As mentioned above, cytokines are also believed to play an important role in a variety of cancers. In the context of prostate cancer, researchers have shown IL-6 to be an autocrine/paracrine growth factor (Seigall et al., supra), to enhance survival of tumors (Okamoto et al. (Cancer Res. 1997), supra), and that neutralizing IL-6 antibodies reduce cell proliferation (Okomoto et al. (Endocrinology 1997), supra; Borsellino et al., supra). IL-6 has also been reported to play a role with regard to multiple myeloma (Martinez-Maza et al., supra; Kawano et al., supra; Zhang et al., supra; Garrett et al., supra; and Klein et al., supra), renal cell carcinoma (Koo et al., supra; Tsukamoto et al., supra; and Weissglas et al., supra), and cervical carcinoma (Estuce et al., supra; Tartour et al., supra; and Iglesias et al., supra).

IL-6 is also believed to be involved in arthritis, particularly in adjuvant-, collagen- and antigen-induced arthritis (Alonzi et al., supra; Ohshima et al., supra; and Leisten et al., supra), and anti-IL-6 antibodies have been reported for treatment of arthritis (Wendling et al., supra). In addition, estrogen has been shown to induce suppression of experimental autoimmune encephalomyelitis and collagen-induced arthritis in mice (Jansson et al., supra).

Accordingly, other embodiments of the present invention include methods for treating bone-resorbing diseases generally, including (but not limited to) osteoporosis, metastatic bone cancer and hypercalcemia, osteolytic lesions with orthopedic implants, Paget's disease, and bone loss associated with hyperparathyroidism. Other conditions associated with IL-6 include various cancers and arthritis. Representative cancers are breast cancer, prostrate cancer, colon cancer, endometrial cancer, multiply myeloma, renal cell carcinoma, and cervical carcinoma. Arthritic conditions include adjuvant-, collagen-, bacterial- and antigen-induced arthritis, particularly rheumatoid arthritis.

In addition, the compounds of the present invention also act as estrogen antagonists and/or agonists, and have utility in the treatment of a wide range of estrogen-related conditions. In this context, treatment includes both treatment and/or prevention of an estrogen-related condition. Thus, the compounds of this invention may be administered as a therapeutic and/or prophylactic agent. An estrogen “agonist” is a compound that binds to ER and mimics the action of estrogen in one or more tissues, while an “antagonist” binds to ER and blocks the action of estrogen in one or more tissues. Further, the term “estrogen-related condition” encompasses any condition associated with elevated or depressed levels of estrogen, a selective estrogen receptor modulators (SERM) or ER. In this context, ER includes both ER-α and/or ER-β, as well as any isoforms, mutations and proteins with significant homology to ER.

Accordingly, the compounds of the present invention may also be used within a method for treating estrogen-related conditions, including (but not limited to) breast cancer, osteoporosis, endometriosis, cardiovascular disease, hypercholesterolemia, prostatic hypertrophy, prostatic carcinomas, obesity, hot flashes, skin effects, mood swings, memory loss, prostate cancer, menopausal syndromes, hair loss (alopecia), type-II diabetes, Alzheimer's disease, urinary incontinence, GI tract conditions, spermatogenesis, vascular protection after injury, endometriosis, learning and memory, CNS effects, plasma lipid levels, acne, cataracts, hirsutism, other solid cancers (such as colon, lung, ovarian, melanoma, CNS, and renal), multiple myeloma, lymphoma, and adverse reproductive effects associated with exposure to environmental chemicals or natural hormonal imbalances.

Compounds of the present invention that are estrogen agonists are useful for oral contraception; relief for the symptoms of menopause; prevention of threatened or habitual abortion; relief of dysmenorrhea; relief of dysfunctional uterine bleeding; relief of endometriosis; an aid in ovarian development; treatment of acne; diminution of excessive growth of body hair in women (hirsutism); the prevention and treatment of cardiovascular disease; prevention and treatment of atherosclerosis; prevention and treatment of osteoporosis; treatment of benign prostatic hyperplasia and prostatic carcinoma obesity; and suppression of post-partum lactation. These agents also have a beneficial effect on plasma lipid levels and as such are useful in treating and preventing hypercholesterolemia. Those which are estrogen antagonists are useful as antiestrogens in, for example, breast and ovarian tissue and thus are useful in the treatment and prevention of breast and ovarian cancer.

Methods of this invention involve administering a compound of structure (I), or a pharmaceutical composition containing the one or more of the same, to an animal in need thereof in an amount sufficient to treat the disease or condition of interest. To that end, the term “treat” (or the related terms “treating” and treatment”) means administration of a compound, typically in combination with an appropriate delivery vehicle or agent, to an animal that does not show signs of a disease or condition (e.g. prophylactic or preventative administration) or that does show signs of a disease or condition (e.g., curative or treatment administration). Further, the phrase “effective amount” means a dose of compound that, after a given time, results in the desired effect. For example, in the context of bone-resorbing disease, an effective amount results in bones mass that is statistically different from that of animals treated with placebo. Similarly, for cancer and arthritis, an effective amount is an amount sufficient to produce the desired effect on the cancerous or arthritic tissue.

The methods of this invention include administration of an effective amount of a compound of structure (I), or a salt thereof, as the active ingredient. Pharmaceutically acceptable salts of the compounds of structure (I) are typically salts of non-toxic type commonly used, such as salts with organic acids (e.g., formic, acetic, trifluoroacetic, citric, maleic, tartaric, methanesulfonic, benzenesulfonic or toluenesulfonic acids), inorganic acids (e.g., hydrochloric, hydrobromic,. sulfuric or phosphoric acids), and amino acids (e.g., aspartic or glutamic acids). These salts may be prepared by the methods known to chemists of ordinary skill.

The compounds of this invention may be administered to animals (including humans) orally or parenterally in the conventional form of preparations, such as capsules, microcapsules, tablets, granules, powder, troches, pills, suppositories, injections, suspensions and syrups. Suitable formulations may be prepared by methods commonly employed using conventional, organic or inorganic additives, such as an excipient (eg, sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate or calcium carbonate), a binder (e.g., cellulose, methylcellulose, hydroxymethylcellulose, polypropylpyrrolidone, polyvinylpyrrolidone, gelatin, gum arabic, polyethyleneglycol, sucrose or starch), a disintegrator (e.g., starch, carboxymethylcellulose, hydroxypropylstarch, low substituted hydroxypropylcellulose, sodium bicarbonate, calcium phosphate or calcium citrate), a lubricant (e.g., magnesium stearate, light anhydrous silicic acid, talc or sodium lauryl sulfate), a flavoring agent (e.g., citric acid, menthol, glycine or orange powder), a preservative (e.g., sodium benzoate, sodium bisulfite, methylparaben or propylparaben), a stabilizer (e.g., citric acid, sodium citrate or acetic acid), a suspending agent (e.g., methylcellulose, polyvinyl pyrroliclone or aluminum stearate), a dispersing agent (e.g., hydroxypropylmethylcellulose), a diluent (e.g., water), and base wax (e.g., cocoa butter, white petrolatum or polyethylene glycol). The amount of the active ingredient in the medical composition may be at a level that will exercise the desired therapeutical effect; for example, about 0.1 mg to 100 mg in unit dosage for both oral and parenteral administration.

The active ingredient may be usually administered one to four times a day with a unit dosage of 0.1 mg to 100 mg in human patients, but the above dosage may be properly varied depending on the age, body weight and medical condition of the patient and the type of administration. A preferred dose is 0.25 mg to 25 mg in human patients. One dose per day is preferred.

Pharmaceutical chemists will recognize that physiologically active compounds which one or more accessible hydroxy groups are frequently administered in the form of pharmaceutically acceptable esters. The literature concerning such compounds, such as estradiol, provides a number of instances of such esters. It is believed that such esters are metabolically cleaved in the body, and that the actual drug is the hydroxy compound itself. It is known known in pharmaceutical arts to adjust the rate or duration of action of a compound by appropriate choices of ester groups.

To this end, prodrugs are also included within the context of this invention. Prodrugs are any covalently bonded carriers that release a compound of structure (I) in vivo when such prodrug is administered to a patient. Prodrugs are generally prepared by modifying functional groups in a way such that the modification is cleaved, either by routine manipulation or in vivo, yielding the parent compound. Prodrugs include, for example, compounds of this invention wherein the hydroxy group (when R₃═H) is bonded to any group that, when administered to a patient, cleaves to form the hydroxy group.

The pharmaceutically acceptable acid addition salts of the compounds of this invention may be formed of the compound itself, or of any of its esters, and include the pharmaceutically acceptable salts which are often used in pharmaceutical chemistry. For example, salts may be formed with inorganic or organic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfonic acids including such agents as naphthalenesulfonic, methanesulfonic and toluenesulfonic acids, sulfuric acid, nitric acid, phosphoric acid, tartaric acid, pyrosulfuric acid, metaphosphoric acid, succinic acid, formic acid, phthalic acid, lactic acid and the like, most preferable with hydrochloric acid, citric acid, benzoic acid, maleic acid, acetic acid and propionic acid. It is usually preferred to administer a compound of this invention in the form of an acid addition salt, as it is customary in the administration of pharmaceuticals bearing a basic group.

The compounds of this invention, as discussed above, are very often administered in the form of acid addition salts. The salts are conveniently formed, as is usual in organic chemistry, by reacting the compound of this invention with a suitable acid, such as have been described above. The salts are quickly formed in high yields at moderate temperatures, and often are prepared by merely isolating the compound from a suitable acidic wash in the final step of the synthesis. The salt-forming acid is dissolved in an appropriate organic solvent, or aqueous organic solvent, such as an alkanol, ketone or ester. On the other hand, if the compound of this invention is desired in the free base form, it is isolated from a basic final wash step, according to the usual practice. A typical technique for preparing hydrochlorides is to dissolve the free base in a suitable solvent and dry the solution thoroughly, as over molecular sieves, before bubbling hydrogen chloride gas through it.

The dose of a compound of this invention to be administered to a human is rather widely variable and subject to the judgment of the attending physician. It should be noted that it may be necessary to adjust the dose of a compound when it is administered in the form of a salt, such as a laureate, the salt forming moiety of which has an appreciable molecular weight. The general range of effective administration rates of the compounds is from about 0.05 mg/day to about 100 mg/day. A preferred rate range is from about 0.25 mg/day to 25 mg/day. Of course, it is often practical to administer the daily dose of compound in portions, at various hours of the day. However, in any given case, the amount of compound administered will depend on such factors as the solubility of the active component, the formulation used and the route of administration.

The route of administration of the compounds of this invention is not critical. The compounds are known to be absorbed from the alimentary tract, and so it is usually preferred to administer a compound orally for reasons of convenience. However, the compounds may equally effectively be administered percutaneously, or as suppositories for absorption by the rectum, if desired in a given instance.

The compounds of this invention are usually administered as pharmaceutical compositions which are important and novel embodiments of the invention because of the presence of the compounds. All of the usual types of compositions may be used, including tablets, chewable tablets, capsules, solutions, parenteral solutions, troches, suppositories and suspensions. Compositions are formulated to contain a daily dose, or a convenient fraction of daily dose, in a dosage unit, which may be a single tablet or capsule or convenient volume of a liquid.

Any of the compounds may be readily formulated as tablets, capsules and the like; it is preferable to prepare solutions from water-soluble salts, such as the hydrochloride salt. In general, all of the compositions are prepared according to known methods in pharmaceutical chemistry. Capsules are prepared by mixing the compound with a suitable diluent and filling the proper amount of the mixture in capsules. The usual diluents include inert powdered substances such as starch of many different kinds, powdered cellulose, especially crystalline and microcrystalline cellulose, sugars such as fructose, mannitol and sucrose, grain flours and similar edible powders.

Tablets are prepared by direct compression, by wet granulation, or by dry granulation. Their formulations usually incorporate diluents, binders, lubricants and disintegrators as well as the compound. Typical diluents include, for example, various types of starch, lactose, mannitol, kaolin, calcium phosphate or sulfate, inorganic salts such as sodium chloride and powdered sugar. Powdered cellulose derivatives are also useful. Typical tablet binders are substances such as starch, gelatin and sugars such as lactose, fructose, glucose and the like. Natural and synthetic gums are also convenient, including acacia, alginates, methylcellulose, polyvinylpyrrolidone and the like. Polyethylene glycol, ethylcellulose and waxes can also serve as binders.

A lubricant is typically necessary in a tablet formulation to prevent the tablet and punches from sticking in the die. The lubricant is chosen from such slippery solids as talc, magnesium and calcium stearate, stearic acid and hydrogenated vegetable oils. Tablet disintegrators are substances which swell when—wetted to break up the tablet and release the compound. They include starches, clays, celluloses, algins and gums. More particularly, corn and potato starches, methylcellulose, agar, bentonite, wood cellulose, powdered natural sponge, cation-exchange resins, alginic acid, guar gum, citrus pulp and carboxymethyl cellulose, for example, may be used as well as sodium lauryl sulfate. Tablets are often coated with sugar as a flavor and sealant, or with film-forming protecting agents to modify the dissolution properties of the tablet. The compounds may also be formulated as chewable tablets, by using large amounts of pleasant-tasting substances such as mannitol in the formulation, as is now well—established in the art.

When it is desired to administer a compound as a suppository, the typical bases may be used. Cocoa butter is a traditional suppository base, which may be modified by addition of waxes to raise its melting point slightly. Water-miscible suppository bases comprising, particularly, polyethylene glycols of various molecular weights are in wide use.

The effect of the compounds may be delayed or prolonged by proper formulation. For example, a slowly soluble pellet of the compound may be prepared and incorporated in a tablet or capsule, or as a slow-release implantable device. The technique may be improved by making pellets of several different dissolution rates and filling capsules with a mixture of the pellets. Tablets or capsules may be coated with a film which resists dissolution for a predictable period of time. Even the parenteral preparations may be made long-acting, by dissolving or suspending the compound in oily or emulsified vehicles which allow it to disperse only slowly in the serum.

The following Examples are presented by way of illustration, not limitation.

EXAMPLES

In summary, Examples 1-7 are directed to the synthesis of a representative compounds of this invention. Example 8 discloses a human bone cell co-culture system for assaying compounds for their ability to block production of IL-6. Example 9 discloses the activity of a representative compound of this invention as assayed in the human bone co-culture system of Example 8. Example 9-16 presents further experimental data evidencing activity of a representative compound of this invention.

Example 1

To a mixture of 3-methoxyphenol (50 g, 0.40 mol), 4-hydroxyphenylacetic acid (71 g, 0.46 mol) and ZnCl₂ (174 g, 1.28 mol) was added POCl₃ (100 ml, 1.6 mol). The mixture was stirred at 65° C. for 2 hours, poured into ice water (2 L) and stirred until the ice melted. The clear supernatant was decanted and the residue was rinsed with water (1 L) and partitioned between EtOAc and water. The organic layer was washed with brine, dried (MgSO₄), filtered and concentrated. The resulting oil was purified by chromatography (SiO₂, 20% EtOAc/n-hexane) to provide 2-(4-hydroxybenzylacetone)-5-methoxyphenol (34.1 g, 33% yield) as a white solid; mp 137-140° C.

Example 2

To a mixture of 2-(4-hydroxybenzylacetone)-5-methoxyphenol (10 g, 0.038 mole), NEt₃ (6 ml, 0.042 mole) in CH₂Cl₂ (50 ml) was added triisopropylsilychloride (9 ml, 0.042 mole). The mixture was stirred for 22 hours, concentrated and the residue partitioned between EtOAc and H₂O. The organic layer was washed with NaOH (1N), HCl (1N) and brine. The organic layer was dried (MgSO₄), filtered and concentrated. The residue was treated with n-hexane to provide 2-(4-triisopropylsilyloxybenzylacetone)-5-methoxyphenol (6.2 g, 38% yield) as a white solid; mp 66-68° C.

Example 3

To a mixture of 2-(4-triisopropylsilyloxybenzylacetone)-5-methoxyphenol (4 g, 9.6 mmole), K₂CO₃ (4 g, 29 mmole) in CH₃CN (50 ml) was added phenyl acetylchloride (2.3 ml, 14 mmole). The mixture was stirred at reflux for 22 hrs, poured into H₂O (0° C.) (500 ml) and extracted with EtOAc (2x). The organic layer was dried (MgSO₄), filtered and concentrated. The residue was stirred with Et₂O and the resulting solid was filtered and recrystallized (EtOH) to give 3-phenyl-4-(4-hydroxybenzyl)-7-methoxycoumarin (0.88 g, 15% yield) as a white solid; mp 235-236° C.

Example 4

A mixture of 3-phenyl4-(4-hydroxybenzyl)-7-methoxycoumarin (0.50 g, 1.39 mmoles), K₂CO₃ (0.58 g, 4.18 mmoles), 2-chloroethylpiperdine hydrochloride (0.41 g, 2.22 mmoles) and acetone (50 ml) was heated at reflux for 6 hours. The solvent was concentrated to a solid which was partitioned between EtOAc and H₂O. The organic layer was washed with NaOH (1H), brine, dried (MgSO₄), filtered and concentrated. The residue was stirred with HCl (20% in EtOAc) and the solid filtered to provide 3-phenyl-4-[4-(2-{piperin-1-yl})ethoxy]-benzyl-7-methoxycoumarin (0.57 g, 87% yield); mp 171-172° C.

Example 5

A mixture of 3-phenyl-4-[4-(2-{piperin-1-yl})ethoxy]-benzyl-7-methoxycoumarin (0.10 g, 0.20 mmole), HOAc (glacial) (15 ml) and HBr (48%, 15 ml) was refluxed for 48 hours. The mixture was partitioned between EtOAc (120 ml) and NaOH (1N, 120 ml) and the aqueous layer was washed with EtOAc. The aqueous layer was then acidified (conc. HCl, pH 1-2) and filtered to provide 3-phenyl-4-[4-(2-{piperidin-1-yl})ethoxy]-benzyl-7-hydroxycoumarin (0.88 g, 99% yield); mp 160-161° C.

Example 6

A solution 3-(4-fluorophenyl)-4-[(4-hydroxyphenyl)methyl]-7-methoxy-2H-chromen-2-one (0.27 g, 0.72 imole), in 3 mL of CH₂Cl₂ was treated with 3-hydroxy-1-methylpiperidine (0.42 g, 3.6 mmol), triphenylphosphine (0.94 g, 3.6 mmol), and diethyl azodicarboxylate (0.65 g, 3.6 mmol). The reaction mixture was stirred for 8 hours at 25° C. then concentrated under reduced pressure. The crude product was dissolved in 4 mL of a 1:1 solution of HBr (48%, aqueous) and glacial acetic acid. The resulting solution was warmed at 90° C. for 12 hours. The reaction mixture was concentrated and the resulting residue was neutralized with 10 mL of saturated aqueous NaHCO₃. The aqueous mixture was extracted with CH₂Cl₂ (3×15 mL) and the combined organic layer was dried (MgSO₄) then concentrated under reduced pressure. The product (106 mg, 32%) was isolated following purification by flash chromatography (SiO₂, CH₂Cl₂/MeOH, 10:1).

Alternatively, 3-(4-fluorophenyl)-4-[(4-hydroxyphenyl)methyl]-7-methoxy-2H-chromen-2-one is reacted with one of the following enantiomers (a) or (b) in the presence of PPh3 and diethyl azodicarboxylate (DEAD), followed by HBr/HOAc, to yield the corresponding enantiomeric product.

Example 7 ADDITIONAL REPRESENTATIVE COMPOUNDS

By the procedures set forth herein, the compounds of Table 1 were prepared.

TABLE 1 Representative Compounds

LC/MS No. R₁ R₂ ^(†) R₃ n p (M + H⁺)  1 phenyl

H 2 1 456  2 4-fluorophenyl

H 2 1 474  3 4-chlorophenyl

H 2 1 490  4 4-bromophenyl

H 2 1 533, 535  5 3-chlorophenyl

H 2 1 490  6 2-chlorophenyl

H 2 1 490  7 3-methylphenyl

H 2 1 470  8 2-methylphenyl

H 2 1 470  9 4-methylphenyl

H 2 1 470 10 4-hydroxyphenyl

H 2 1 473 11 3-bromopyridin-3-yl

H 2 1 535, 537 12 3,4-dichlorophenyl

H 2 1 524, 526 13 thiophen-2-yl

H 2 1 462 14 4-trifluoromethylphenyl

H 2 1 524 15 4-chlorophenyl

H 2 2 504 16 4-chlorophenyl

H 2 0 476 17 phenyl

H 2 1 458 18 4-fluorophenyl

H 2 1 474 19 phenyl

SO₂N(CH₃)₂ 2 1 563 20 phenyl

CON(CH₃)₂ 2 1 527 21 phenyl

CO(phenyl) 2 1 560 22 phenyl

COCH₃ 2 1 498 23 phenyl

COOCH₂CH₃ 2 1 528 24 4-fluorophenyl

2 1 585 25 4-fluorophenyl

CH₂CH₂N(CH₃)₂ 2 1 574 26 4-chlorophenyl

H 2 1 490 27 4-chlorophenyl

H 2 1 490 28 4-fluorophenyl

H 1 1 533 29 4-fluorophenyl

H 2 1 457 30 4-fluorophenyl

H 0 1 460 31 4-fluorophenyl

H 1 1 460 32 4-fluorophenyl

H 0 1 460 33 4-chlorophenyl

SO₂NH₂ 2 1 570 34 2,4-difluorophenyl

H 2 1 492 35 2,4-dichlorophenyl

H 2 1 524 36 4-chlorophenyl

SO₂CH₃ 2 1 568 37 4-fluorophenyl

H 3 1 448 38 phenyl

H 2 0 430 ^(†)At the 4-position of the phenyl ring unless otherwise noted ^(‡)At the 3-position of the phenyl ring

Example 8 HUMAN BONE CELL CO-CULTURE SYSTEM

The generation of osteoblastic cells from normal adult femoral travecular bone from patients without evidence of metabolic bone disease was carried out as previously described (Kung Sutherland et al., Osteoporosis International 5:335-343, 1995; Wong et al., J Bone Miner 5:803-813, 1990). Bone fragments were cleaned of adherent tissue and blood and cultured for 4 weeks in medium containing Ham's F12 supplemented with 28 mM HEPES, pH 7.4, 10% FCS, 1.1 MM CaCl₂, 2 mM glutamine, and 1% antibiotic-antimycotic agent. Upon confluency, the cells were harvested and immortalized using a retroviral vector (pLXSN) containing the neomycin resistance gene and the human papillomaviral (HPV18) E6 and E7 concogenes (International Journal of Oncology 6:167-174, 1995). The immortalized osteoblastic cells were selected in medium containing G418 (600 μg/ml).

The human U937 moncytic cell line (cloned from the commercially available cells by serial dilution) was maintained in RPMI 1640 medium containing 10% FCS and 1% antibiotic-antimycotic agent.

The co-culture system was established as follows. Human osteoblastic cells were plated in 96well dishes at a density of 5×10³ cells/well. U937 cells in RPMI medium (5×10⁴ cells/well) were layered over the osteoblasts. The formation of osteoclast-like cells was induced with 100 nM PMA. To determine the effects of compounds or the neutralizing antibodies to IL-6 (Sigma) and GM-CSF (Pharmingen) on the formation of osteoclast-like cells, the compounds or the antibodies were added 30 minutes prior to the addition of PMA. The cultures were stopped after 96 hrs and processed for histochemical or immunohistochemical analyses.

Example 9 ACTIVITY OF REPRESENTATIVE COMPOUND IN THE HUMAN BONE CO-CULTURE

The compound of Example 5 (i.e., Compound No. 1 of Table 1) was assayed in the human bone co-culture system of Example 8 and found to have an IC₅₀ with respect to IL-6 and GM-CSF of 10 nM and 38 nM, respectively.

Example 10 ACTIVITY OF REPRESENTATIVE COMPOUND ON IL-6 AND GM-CSF PRODUCTION IN HOB CELLS

Human osteoblasts (HOB) were plated in 96-well dishes at a density of 7×10³ cells/well in regular HOB medium (Ham's F12 supplemented with 28 mM HEPES, pH 7.4, 10% FCS, 1.1 mM CaCl₂, 2 mM glutamine, and 1% antibiotic-antimycotic agent). The following day, the cells were treated with Compound 1 or vehicle (0.2% DMSO) for 30 minutes prior to the addition of the combination of IL-1β (1 ng/ml) and TNF-α (10 ng/ml). Cultures were continued for 18 to 24 hrs. IL-6 and GM-CSF levels in culture medium were measured using commercially available ELISA kits (Endogen, Cambridge, Mass.). The results of this experiment are set forth in FIG. 1A for IL-6 inhibition, and FIG. 1B for GM-CSF inhibition. These figures also present the comparative data for diethylstilbestrol (DES) and Raloxifene.

Example 11 ACTIVITY OF REPRESENTATIVE COMPOUND ON IL-6 PRODUCTION IN RA CELLS

Primary rheumatoid arthritic synoviocytes were plated in 96-well dishes at a density of 8×10³ cells/well in phenol-red free DMEM supplemented with 2 mM glutamine, 1% antibiotic-antimycocit agent and 10% charcoal-stripped FCS. The following day, the cells were treated with Compound No. 1 or vehicle (0.2% DMSO) for 30 minutes prior to the addition of the combination of IL-1β (2 ng/ml) and TNF-α (2 ng/ml). Cultures were continued for 18 to 24 hrs. IL-6 levels in culture medium were measured using commercially available Endogen ELISAs as disclosed above. The results of this experiment are presented in FIG. 2, as well as the comparative activity at DES and Raloxifene.

Example 12 ACTIVITY OF REPRESENTATVE COMPOUND ON PROLIFERATION OF BREAST CANCER CELLS

MCF-7 breast carcinoma cells were plated in 24-well dishes at a density of 5×10³ cells/well in phenol-red free DMEM:F-12 (1:1) medium containing 1% antibiotics, 0.05% β-mercaptoethanol, 0.01% ethanolamine, 0.42 ng/ml sodium selenite and 5% charcoal-stripped FCS. Compound No. 1 and vehicle (0.2% DMSO) were added the following day and refreshed with media change every 48 hours. Cultures were stopped 9 days later and proliferation assayed using the Cyquant kit (Molecular Probes, Eugene, Oreg.). The results of this experiment are presented in FIG. 3, including comparative data for Tamoxifen and Raloxifene, as well as for the combination of the same with Estradiol.

Example 13 ACTIVITY OF REPRESENTATIVE COMPOUND ON PROLIFERATION OF PROSTATE CARCINOMA CELLS

DU-145 prostate carcinoma cells were plated in 96-well dishes at a density of 2×10³ cells/well in phenol-red free MEM Eagles medium containing Earles' Balanced salts, 1% antibiotic-antimycotic agent, 2 mM glutamine, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, and 10% charcoal-stripped FCS. Compound No. 1 and vehicle (0.2% DMSO) were added the following day and refreshed with medium change every 48 hrs. Cultures were stopped 5 days later and proliferation assayed using the Cyquant kit as disclosed above. The results of this experiment are presented in FIG. 4, with comparative data for DES.

EXAMPLE 14 SOLID PHASE ER BINDING ASSAY

Binding to human Estrogen Receptor (ER-α and ER-β) was evaluated in a solid-phase ³H-estradiol competition assay, as adapted from McGuire, Cancer Res. 38:4289-4291, 1978. Briefly, human recombinant ER-α (15 nM) or ER-β (50 nM) was immobilized to wells of 96-well microtiter plates. Binding of test compound to ER was evaluated in competition with 30 nM ³H-estradiol at room temperature for 1 hour. The results of this experiment, as presented in Table 2, represent the average from at least 3 different experiments. Reference compounds were tested concurrently as an integral part of each assay to ensure the validity of the results obtained.

TABLE 2 Solid Phase ER Binding K_(i) (nM) Receptor Compound No. 1 Tamoxifen Assay (HCl salt) citrate Raloxifene HCl Estradiol ER-α 1.4  72 0.4 0.8 ER-β 7.3 173 13 2.5

Binding data for tamnoxifen, raloxifene, and estradiol were found to correspond to published literature values for these compounds. Binding of Compound No. 1 (in the form of the HCl salt) to ER-α and ER-β was compared to binding of 17β-estradiol, tamoxifen citrate and raloxifene.HCl. Compound No. 1 binds with high affinity (similar to estradiol) to ER-α and ER-β. Affinity for ER-β was found to be slightly lower than from ER-α, as is typical for most ER ligands

Example 15 UTERINE LIABILITY

As discussed herein, compounds that function as Selective Estrogen Receptor Modulators or SERMs are desirable. In a preferred embodiment, SERMs do not exhibit uterine liability as assessed in recognized assays, such as the immarture rat uterine bioassay (Robertson et al., Biol. Reprod. 54:183-196, 1996; Medlock et al., Biol. Reprod. 56:1239-1244, 1997; Martel et al., Endocrinology 139:2486-2492, 1998; Hyder et al., Cancer Cells 120:165-171, 1997), and the ovariectomized rat uterine bioassay (Sato et al., FASEB J. 10905-912, 1996; Ruenitz et al., Bone 23:537-542, 1998; Luo et al., Endocrinology 139:2645-2656, 1998; Ke et al., Bone 20:31-39, 1997). Both of these assays measure the effect of a particular compound on uterine wet and dry weight, and provide histological data.

Compound No. 1 was tested side-by-side with estrogen and Raloxifene.HCl in a 3-day immature rat uterine biassay using subcutaneous (s.c.) administration (Medlock et la., supra). Estrogen caused a 4.5-fold increase in uterine weight, which is the reported literature value (Ashby et al., Regul. Toxicol. Pharmacol. 25:226-231, 1997; Medlock et al., supra). Raloxifene.HCl caused up to 50% increase in uterine weight at the 1 mg/kg dose, which is also in agreement with published data (Ashby et al., supra). Compound No. 1 caused about a 30% increase in uterine weight, which was not statistically different from the vehicle-treated animals (i.e., PEG-400). Both RaloxifeneHCl and Compound No. 1 displayed a bell-shaped dose response, which is typically found with SERMs.

Compond No. 1 was also tested side-by-side with 17β-estradiol and Raloxifene.HCl in a 28-day uterine bioassay in ovariectomized rats (Ke et al., supra). All three componds were administered daily by s.c. injection. Estrogen prevented the uterine weight loss caused by ovariectomy by increasing the uterine weight by 550%. These results are in agreement with published data (Grese et al., Proc. Natl. Acad. Sci. USA 94:14105-14110, 1997; Ashby et al., supra; Ke et al., supra). Raloxifene.HCl caused about a 70% increase in uterine weight which has been previously reported (Grese et al., supra; Ashby et al., supra). Compound No. 1 caused about a 40% increase in uterine weight, which was statistically less than the Raloxifene.HCl-mediated weight increase. These results support the data from the immature rat uterine bioassay that Compound No. 1 does not exhibit significant uterine liability. Thus, this compound has significant potential for the prevention and/or treatment of cancer and osteoporosis, as well as cardiovascular diseases and neurodegenerative diseases such as Alzheimer's disease.

EXAMPLE 16 COMPARATIVE TESTING

Compound No. 1 was tested against a compound of Lednicer et al., J. Med. Chem. 8:725-726, 1965. More specifically, this reference is directed to coumarin-based compounds as purported estrogen antagonists. One of these compounds (ie., Compound 20 of Table IV, hereinafter “L-20”) has the following structure:

The above compound was tested against Compound No. 38 of the present invention in the ER binding assay of Example 14 and the human bone co-culture system of Example 8 for IL-6 activity. The results of these experiments are presented in Table 3.

TABLE 3 ER Binding Assay IL-6 Activity ER-α(K_(i), μM) ER-β(K_(i), μM) (IC₅₀, μM) Compound No. 38 0.69 1.56 0.175 “L-20” >10 >10 >1

It will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without departing from the spirit and scope of the invention. Accordingly, the invention is not limited except by the following claims. 

What is claimed is:
 1. A compound having the structure:

or a stereoisomer, prodrug or pharmaceutically acceptable salt thereof, wherein: n is 0, 1, 2, 3 or 4; p is 1 or 2; R₁ is an unsubstituted or substituted C₆₋₁₂aryl, C₇₋₁₂arylalky, C₃₋₁₂heterocycle or C₄₋₁₆heterocyclealkyl; R₂ is NR_(a)R_(b) wherein R_(a) and R_(b) are independently hydrogen, C₁₋₈alkyl, C₆₋₁₂aryl, or heterocycle, and wherein R_(a) and R_(b) are optionally substituted with up to three substituents independently selected from C₁₋₆alkyl, halogen, C₁₋₆alkoxy, hydroxy and carboxyl; or R₂ is a heterocyclic ring of the following structure:

wherein m₁ and m₂ are independently 0, 1 or 2, and both m₁ and m2 are not 0, A is CH₂, O, S or NH, Z represents 0, 1, 2 or 3 heterocyclic ring substituents selected from halogen, C₁₋₈alkyl, C₆₋₁₂aryl, C₇₋₁₂arylalky, C₃₋₁₂heterocycle, or C₄₋₁₆heterocyclealkyl, and wherein any hydrogen atom on the heterocyclic ring may, taken together with a hydrogen atom on an adjacent atom of the heterocylic ring, form a double bond; R₃ is hydrogen, R₄, C(═O)R₄, C(═O)OR₄, CONHR₄, CONR₄R₅, or SO₂NR₅R₅; R₄ and R₅ are independently C₁₋₈alkyl, C₆₋₁₂aryl, C₇₋₁₂aralkyl, or a five- or six-membered heterocycle containing up to two heteroatoms selected from O, NR₆ and S(O)_(q), wherein each of the above groups are optionally substituted with one to three substituents independently selected from R₇ and q is 0, 1 or 2; R₆ is hydrogen or C₁₋₄alkyl; and R₇ is hydrogen, halogen, hydroxy, C₁₋₆alkyl, C₁₋₄alkoxy, C₁₋₄acyloxy, C₁₋₄thio, C₁₋₄alkylsulfinyl, C₁₋₄alkylsulfonyl, (hydroxy)C₁₋₄alkyl, C₆₋₁₂aryl, C₇₋₁₂aralkyl, COOH, CN, CONHOR₈, SO₂NHR₈, NH₂, C₁₋₄alkylamino, C₁₋₄dialkylamino, NHSO₂R₈, NO₂, or a five- or six-membered heterocycle, where each occurrence of R₈ is independently C₁₋₆alkyl.
 2. The compound of claim 1 wherein R₁ is an unsubstituted or substituted C₆₋₁₂aryl.
 3. The compound of claim 2 wherein R₁ is an unsubstituted or substituted phenyl.
 4. The compound of claim 3 wherein R₁ is unsubstituted phenyl.
 5. The compound of claim 3 wherein R₁ is substituted phenyl.
 6. The compound of claim 5 wherein R₁ is 4-halophenyl, 4-methylphenyl, 4hydroxyphenyl, 4-trifluoromethylphenyl, 3-halophenyl, 2-halophenyl, 2,4-dihalophenyl, or 3,4-dihalophenyl, wherein halo is fluoro, chloro, bromo or iodo.
 7. The compound of claim 1 wherein R₁ is an unsubstituted or substituted heteroaryl.
 8. The compound of claim 7 wherein R₁ is substituted or unsubstituted pyridinyl or thiophenyl.
 9. The compound of claim 1 wherein R₁ is an unsubstituted or substituted C₇₋₁₂arylalkyl.
 10. The compound of claim 9 wherein R₁ is an unsubstituted or substituted benzyl.
 11. The compound of claim 1 wherein R₁ is an unsubsituted or substituted C₃₋₁₂heterocycle or C₄₋₁₆heterocyclealkyl.
 12. The compound of claim 1 wherein n is
 2. 13. The compound of claim 1 wherein n is 0, 1, 3 or
 4. 14. The compound of claim 1 wherein p is
 1. 15. The compound of claim 1 wherein p is
 2. 16. The compound of claim 1 wherein R₃ is hydrogen.
 17. The compound of claim 1 wherein R₃ is C(═O)(C₁₋₈alkyl) or C(═O)(C₆₋₁₂aryl).
 18. The compound of claim 1 wherein R₃ is C(═O)O(C₁₋₈alkyl), SO₂NH₂ or CONH₂.
 19. The compound of claim 1 wherein R₂ is a heterocyclic ring of the following structure:


20. The compound of claim 19 wherein m₁ and m₂ are
 1. 21. The compound of claim 20 wherein A is CH₂.
 22. The compound of claim 21 wherein R₂ is piperdin-1-yl.
 23. The compound of claim 20 wherein A is O.
 24. The compound of claim 23 wherein R₂ is morpholin-4-yl.
 25. The compound of claim 19 wherein m₁ is 1 and m₂ is
 0. 26. The compound of claim 25 wherein A is CH₂ and R₂ is imidazolidin-2-yl substituted with 0 or 1 Z substituents.
 27. The compound of claim 19 wherein R₂ is imidazol-1-yl or imidazol-2-yl substituted with 0 or 1 Z substituents.
 28. The compound of claim 1 wherein the R₂—(CH₂)_(n)—O— moiety is attached at the 4-position of the phenyl ring.
 29. The compound of claim 1 wherein the R₂—(CH₂)_(n)—O— moiety is attached at the 3-position of the phenyl ring.
 30. The compound of claim 1 having the structure:


31. The compound of claim 1 having the structure:

wherein X represents one or more phenyl substituents.
 32. The compound of claim 30 having the structure:


33. The compound of claim 1 having the structure:

wherein: (a) each X is independently halogen or trifluoromethyl and (b) X is present at either the 2- and 4-position or the 4-position of the phenyl ring.
 34. The compound of claim 33 having the structure:

wherein X is halogen.
 35. A pharmaceutical composition comprising a compound of claim 1 in combination with a pharmaceutically acceptable carrier or diluent.
 36. A method for inhibiting a cytokine in an animal in need thereof, comprising administering to the animal an effective amount of the composition of claim
 35. 37. The method of claim 36 wherein the cytokine is IL-6.
 38. The method of claim 36 wherein the cytokine is GM-CSF.
 39. A method for treating a bone-resorbing disease in an animal in need thereof, comprising administering to the animal an effective amount of the composition of claim
 35. 40. The method of claim 39 wherein the bone-resorbing disease is osteoporosis.
 41. The method of claim 39 wherein the bone-resorbing disease is metastatic bone cancer, osteolytic lesions with an orthopedic implant, Paget's disease, or bone loss associated with hyperparathyroidism.
 42. A method for treating cancer associated with IL-6 in an animal in need thereof, comprising administering to the animal an effective amount of the composition of claim
 35. 43. The method of claim 42 wherein the cancer is breast cancer, prostrate cancer, colon cancer, endometrial cancer, multiple myeloma, renal cell carcinoma, or cervical carcinoma.
 44. A method for treating arthritis in an animal in need thereof, comprising administering to the animal an effective amount of the composition of claim
 35. 45. The method of claim 44 wherein the arthritis is rheumatoid arthritis.
 46. A method for modulating gene expression in a cell expressing ER, comprising contacting the cell with an effective amount of a compound of claim
 1. 47. The method of claim 46 wherein ER is ER-α.
 48. The method of claim 46 wherein the cell is of bone, bladder, uterus, ovary, prostate, testis, epididymis, gastrointestinal tract, kidney, breast, eye, heart, vessel wall, immune system, lung, pituitary, hippocampus or hypothalamus.
 49. A method for modulating ER in tissue expressing ER, comprising contacting the tissue with an effective amount of a compound of claim
 1. 50. The method of claim 49 wherein ER is ER-α.
 51. The method of claim 49 wherein the tissue is of bone, bladder, uterus, ovary, prostate, testis, epididymis, gastrointestinal tract, kidney, breast, eye, heart, vessel wall, immune system, lung, pituitary, hippocampus or hypothalamus.
 52. A method for treating an estrogen-related condition, comprising administering to an animal in need thereof an effective amount of a pharmaceutical composition of claim
 35. 53. The method of claim 52 wherein the estrogen-related condition is breast cancer, osteoporosis, endometriosis, cardiovascular disease, hypercholesterolemia, prostatic hypertrophy, prostatic carcinomas, obesity, cataracts, hot flashes, skin effects, mood swings, memory loss, prostate cancer, menopausal syndromes, type-II diabetes, Alzheimer's disease, urinary incontinence, GI tract conditions, spermatogenesis, vascular protection after injury, endometriosis, learning and memory, CNS effects, plasma lipid levels, acne, hirsutism, solid cancers, multiple myeloma, lymphoma, or adverse reproductive effects associated with exposure to environmental chemicals or natural hormonal imbalances.
 54. The method of claim 46 where ER is ER-β.
 55. The method of claim 49 wherein ER is ER-β.
 56. The compound of claim 33 wherein the halogen is fluorine or chlorine.
 57. The method of claim 36 wherein the compound has the structure:

wherein: (a) each X is indepemdently halogen or trifluoromethyl and (b) X is present at either the 2- and 4-position or the 4-position of the phenyl ring.
 58. The method of claim 57 wherein the halogen is fluorine or chlorine.
 59. The method of claim 39 wherein the compound has the structure:

wherein: (a) each X is independently halogen or trifluoromethyl and (b) X is present at either the 2- and 4-position or the 4-position of the phenyl ring.
 60. The method of claim 59 wherein the halogen is fluorine or chlorine.
 61. The method of claim 42 wherein the compound has the structure:

wherein: (a) each X is independently halogen or trifluoromethyl and (b) X is present at either the 2- and 4-position or the 4-position of the phenyl ring.
 62. The method of claim 61 wherein the halogen is fluorine or chlorine.
 63. The method of claim 44 wherein the compound has the structure:

wherein: (a) each X is independently halogen or trifluoromethyl and (b) X is present at either the 2- and 4-position or the 4-position of the phenyl ring.
 64. The method of claim 63 wherein the halogen is fluorine or chlorine.
 65. The method of claim 46 wherein the compound has the structure:

wherein: (a) each X is independently halogen or trifluoromethyl and (b) X is present at either the 2- and 4-position or the 4-position of the phenyl ring.
 66. The method of claim 65 wherein the halogen is fluorine or chlorine.
 67. The method of claim 49 wherein the compound has the structure:

wherein: (a) each X is independently halogen or trifluoromethyl and (b) X is present at either the 2- and 4-position or tle 4-position of the phenyl ring.
 68. The method of claim 67 wherein the halogen is fluorine or chlorine.
 69. The method of claim 52 wherein the compound has the structure:

wherein: (a) each X is independently halogen or trifluoromethyl and (b) X is present at either the 2- and 4-position or the 4-position of the phenyl ring.
 70. The method of claim 69 wherein the halogen is fluorine or chlorine.
 71. The compound of claim 33 wherein the group represented by


72. The method of claim 57 wherein the group represented by


73. The method of claim 59 wherein the group represented by


74. The method of claim 61 wherein the group represented by


75. The method of claim 63 wherein the group represented by


76. The method of claim 65 wherein the group represented by


77. The method of claim 67 wherein the group represented by


78. The method of claim 69 wherein the group represented by 